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Single cell characterization of arrested B-lymphoid differentiation and leukemic cell states in ETV6-RUNX1-positive pediatric leukemia

ABSTRACT: Genetic hits in the gene regulatory network underlie arrested lymphoid cell differentiation and emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). Here, we aimed to understand what alterations in the lymphoid gene expression program arise in leukemias carrying the common translocation t12;21, fusing the ETV6 and RUNX1 genes. We compared normal B-lineage differentiation and in vivo leukemic cell states at diagnosis and during chemotherapy using single cell RNA-sequencing (scRNA-seq) and several complementary genomics assays. We show that noisy gene expression of critical pre-BCR signaling pathway genes arises as a consequence of ETV6-RUNX1-mediated repression of intronic enhancers. Furthermore, our analysis implicated the genome-wide association study hit ELK3 among high activity transcription factors in leukemic cell states. The accompanying gene expression changes were found to associate with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes represent features of chemoresistance during induction chemotherapy. Finally, we show that selective inhibitors of ETS-transcription factors could dramatically reduce cell viability. In summary, our results provide a detailed picture of gene regulatory defects in leukemic bone marrow, disclosing mechanistic insight on the role of gene expression noise and suggesting new treatment strategies targeting the active regulatory network.

PROVIDER: EGAS00001004374 | EGA |

REPOSITORIES: EGA

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Project description:Background: ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation, whereas its role in leukemia propagation and maintenance remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines. Findings: Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-DOWN set was significantly enriched for genes included in the cell activation, immune response, apoptosis, signal transduction and development and differentiation categories, whereas in the E/R KD-UP set only the PI3K/AKT/mTOR signaling and hematopoietic stem cells categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories stem cell properties, B-cell differentiation, immune response, cell adhesion and DNA damage with RT-qPCR. Conclusion: The results of our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with regular B-cell development by repressing key functions that are necessary under physiological circumstances. E/R may thus constitute also the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets. Knockdown of ETV6/RUNX1 in two t(12;21) positive leukemic cell lines

Project description:ETV6-RUNX1 is associated with childhood B-cell acute lymphoblastic leukemia (B-ALL), but the consequence of ETV6-RUNX1 expression on cell lineage decision during B-cell leukemogenesis remains largely evasive. Clinically silent ETV6-RUNX1 preleukemic clones are frequently found in neonatal cord blood, but only a few carriers develop B-ALL as a result of the acquisition of secondary postnatal genetic hits, like KDM5C or PAX5 loss. However, understanding the mechanism involved in this transformation would advance the development of non-toxic prophylactic interventions to preleukemic carriers. Using genetic lineage tracing, we have examined the capacity of ETV6-RUNX1 in instructing a B-cell malignant phenotype. Restricted Cre-mediated activation of ETV6-RUNX1 from the endogenous Etv6 locus into B-cell precursors does not trigger B-ALL development. Similarly, concomitant transient ETV6-RUNX1 expression in hematopoietic progenitors close to restricted Cre-mediated introduction of second hit (Kdm5c loss) in B-cells fail to induce B-ALL. In contrast, targeting ETV6-RUNX1 in hematopoietic progenitors was required to induce leukemia. These mice develop either T-ALL or B-ALL, suggesting that the nature of the second hit may define tumor cell identity during ETV6-RUNX1 leukemogenesis. In order to uncover a potential exclusive effect of the second hit in establishing the leukemic phenotype, we next showed that the introduction of the second hit, either Kdm5c or Pax5 loss, to the ETV6-RUNX1 preleukemic clone confers the tumor B-cell identity without need of environmental infection exposure. The resulting B-ALLs clustered according to the second-hit by RNA sequencing (RNA-Seq) analysis. Together these results provide a novel paradigm for the generation of tumor B cells through ETV6-RUNX1 in vivo where the second hit confers the tumor cell-identity to the ETV6-RUNX1 preleukemic clone. These findings could be relevant to develop new therapeutic approaches to prevent disease development.

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Single cell characterization of arrested B-lymphoid differentiation and leukemic cell states in ETV6-RUNX1-positive pediatric leukemia

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